To more completely assess the fungal species to which people are exposed, a fungal ribosomal RNA (rRNA) gene sequencing study was designed to test the hypothesis that fungal bioaerosols in the United States indoor built environments are much more diverse than previously estimated using traditional methods of analysis. Fungal rRNA sequencing data from samples collected from a variety of geographic regions within the United States including Atlanta (Georgia), Bennington (Vermont), Reno (Nevada) and Philadelphia (Pennsylvania) were analyzed using Illumina miSeq high throughput sequencing or Sanger sequencing to characterize fungal diversity in air and dust samples. In FY17 the project was extended to include the analysis of fungal diversity in occupational environmental samples collected in NIOSH Health Hazard Evaluations (HHEs). Analysis of area and personal air samples collected in the cannabis production industry has demonstrated a diversity of fungi primarily composed of species placed in the phylum Ascomycota including plant pathogenic species such as Botrytis cinerea, which causes grey mold. Other plant pathogens placed in the phylum Basidiomycota, including Wallemia sebi have also been enumerated in another recently sampled production facility. These HHEs have highlighted that workers can also be exposed to microbial contaminants including species that have been previously associated with occupational cases of hypersensitivity pneumonitis and allergic sensitization. The results of ITS rRNA gene sequencing studies have also provided new insights into the spectrum of fungi in occupational environments previously overlooked using traditional methods of assessment. Additional studies are examining the molecular diversity of fungal contaminants as part of the NYC Neighborhood Asthma and Allergy Study. Preliminary analysis of analyzed dust samples indicates that Aureobasidium pullulans, Penicillium glabrum, Wallemia sebi and Alternaria alternata varied by housing type (single, multi-family or apartment) and neighborhood asthma prevalence. Preliminary results suggest that multiple environmental factors including anthropogenic behavior modification, housing type, and neighborhood are important variables that influence fungal diversity within middle-income homes in New York City. A. alternata measured in house dust was also associated with fractional exhaled nitric oxide, specifically among children with higher combustion byproduct exposure, suggesting a possible interaction between these two exposures on airway inflammation. Fungal diversity captured using Illumina miSeq is being compared to results obtained using qPCR and the data analysis is ongoing. Work has continued in the development of antibodies to recombinant fungal biomarker antigens. The utility of these antibodies is critical for the quantification of fungal biomarkers, particularly to those fungi that are being studied by the NTP. NIOSH has recently developed a recombinant Alt a 1 homologue from Ulocladium chartarum tentatively named Ulo c 1. Polyclonal antibodies (pAb) have been produced toward the recombinant Ulo c 1. In FY17, pAb reactivity with rUlo c 1 as well as cross-reactivity with other Alt a 1 homologs has been determined and sandwich and inhibition ELISAs developed. Epitope mapping experiments and optimization of the immunoassay will continue in FY18.